Method and device for analyzing nucleic acids immobilized on a support

ABSTRACT

A process for characterizing, quantifying or mapping a nucleic acid or a nitrogenous base fixed on a support by using a photothermal (mirage effect) method. An improved process for manufacturing a nucleic acid biochip on which at least one nucleic acid synthesized in situ is fixed. A device for implementing these processes.

TECHNICAL FIELD

This invention particularly relates to a process for characterization,quantification and mapping of a nitrogenous base, a nucleic acid or anitrogenous base of a nucleic acid fixed on a support. It also relatesto a process for manufacturing a biochip with nucleic acids formedparticularly on a solid support on which at least one nucleic acid isfixed, and a device for implementing the process according to thisinvention.

In general, this invention is used for applications in the fields ofchemical or biological analysis systems used for sequencing and studyingexpression of genes.

For example, these devices may be composed of a set of identical ordifferent molecular probes, for example of nucleic acids fixed on theminiaturized surface of a support or a micro surface. They form what isnormally called a biochip, or a DNA chip when the nucleic acid is adeoxyribonucleic acid and an RNA chip when the nucleic acid is aribonucleic acid. All nucleic acids fixed on the support micro-surfacesform a probes matrix.

During the analysis of a sample using a DNA chip, the target nucleicacids of an extract are marked and deposited on the probes matrix.Hybridisation, in other words matching between molecules ofcomplementary nucleic acids, between probes and marked targets, is usedto mark and identify sequences of nucleic acids present in the analysedsample.

Many processes for the manufacture of biochips have been described anddeveloped in recent years to improve miniaturization and the capacity ordensity of analysis sites on a chip.

Some of these consist of an in situ synthesis of probe nucleic acids onstructured substrates. The synthesis method makes use of two differentaddressing modes to structure the substrate; either a manual addressingmode or a mechanical addressing mode, or a photochemical addressing modeor a mode using lithography techniques.

The first addressing mode is manual addressing using a micro robot orusing an automatic synthesiser coupled to the substrate structure. Forexample, this addressing mode is disclosed in document Southern EM,Nucleic Acid research, Apr. 25, 1994, 22(8): 1368–1373.

A significant improvement to deposition techniques by micro-pipettingusing micro robots or jet printing methods makes it possible to envisageindustrial processes for the production of probes using chemicalsynthesis methods. Document WO-A-94 27719 by PROTOGENE LAB INC. and A.P. Blanchard, R. J Kaiser, L. E Hood, Biosensors and Bioelectronics, vol11, No. 6/7, pages 687 to 690, 1996 discloses use of jet printingtechniques to distribute the four basic activated nucleotides of DNA andthe coupling reagents, on the different sites of the DNA biochip.

The second addressing mode comprising photochemical addressingtechniques and lithography techniques is disclosed for example inAffymetrix, Proc. Natl. Acad. Sci. USA, 1996, Nov. 26; 93(24), 13555–60.

In these two addressing modes, the synthesis uses conventional couplingreactions by means of phosphoramidites or phosphites for successivecondensation of judiciously protected nucleosides. The Caruthersdocument, Science, Oct. 85, page 281, discloses a synthesis cyclecomprising deprotection, coupling, capping and oxidation steps. Thiscycle is a means of making the trace nucleotide grow from the supportsurface forming the biochip.

Unlike some addressing modes in which the trace nucleotide ispresynthesised and therefore purified and qualified before being graftedonto the solid support, the two addressing modes mentioned above requirecharacterization of nucleic acids synthesised after each nucleotidecoupling step, since purification is not possible after synthesis.

For example, a synthesis on a solid support in (100×100) μm² sitesstarting from reagent quantities of a few nl, requires optimisation ofthe synthesis process in order to obtain a coupling efficiency as closeas possible to 100%. The quality of hybridising will depend on thepurity of the synthesised probes. Therefore, it is necessary to qualifyeach synthesis step and to be able to check synthesised sequences.

Furthermore, after carrying out the trace nucleotides synthesis, a checkon the probe density and the uniformity of this density over thesubstrate has to be made.

STATE OF THE ART

The most frequently used method for efficiency calculations for eachstep is to measure the absorption of dimethoxytrityl cations at 500 nmafter deprotection of nucleosides. For example, this method is describedin Tetrahedron Letters, vol. 25, No. 4, pages 375 to 378, 1984.

The absorbance to be measured can vary between 0.2 and 10⁻³ depending onthe quantities of synthesised probes, and this absorption is usuallymeasured by a double beam spectrometer with an absolute precision of theorder of 0.1%. There is a sensitivity problem with the characterizationinstrument necessary for in situ synthesis. If the absorbancemeasurement is sufficiently precise (<10⁻³), the efficiency can becalculated for each step. This measurement is not specific to a base andtherefore cannot give any information about the synthesised sequence.Proportioning of the trityl cation is global for the entire substrateand can be used to calculate an average probe density for eachsubstrate, but it does not give any information about its uniformity.

The document entitled Pease A. C Proc. Natl. Acad. Sci. USA, May 1994,vol. 91, pages 5022 to 5026 discloses a measurement of the fluorescenceafter hybridisation with marked probes equivalent to the synthesisedprobes used to determine the sequences. But this measurement is madeafter the probes have been fully synthesised, and cannot provide anyinformation about the step by step efficiency.

An improvement to this technique has just been proposed by Affymetrix inGlen Mac Gall, J. Org. Chem., 1998, 63, pages 241 to 246. This techniquecan give an efficiency for each step in the synthesis. Regions ofvariable lengths of nucleic acids are defined by lithography and at theend of the synthesis, coupling is done with a phosphoramidite containingfluoresceine on all variable length probes.

This method can be used to develop synthesis processes on a solidsupport, but it cannot characterise the successive steps in graftingtrace nucleotides before the end of the complete synthesis of thedifferent trace nucleotides.

The MALDI-TOF (Matrix Assisted Laser Descrption/Ionization Time ofFlight) technique disclosed in Little P. D., Anal. Chem. 1997, 69, pages4540 to 4546, has been used to analyse biochips containing DNA probes.This is the technique that provides the best resolution at the momentfor analysis quantities of up to 2.5 fentomoles. Unfortunately, it isdestructive and requires a special implementation. Probes must bechemically cleavable at the end of the synthesis and must beco-crystallised with a material capable of absorbing the laser used.

Another technique consists of cleaving the probes after synthesis. Theycan then be analysed by HPLC (High Pressure Liquid Chromatography). Thismethod can help with the development of synthesis processes on a solidsupport, but can never be used for characterisation of in situ synthesison a structured substrate.

DESCRIPTION OF THE INVENTION

The purpose of this invention is to provide a process that is usedparticularly to qualify each step in the synthesis of a biochip and toverify the sequence of nucleic acids synthesised on the support, andtheir density and uniformity, the said process overcoming the abovementioned problems encountered with techniques according to prior art,particularly for checking the manufacture of biochips with nucleicacids.

The process according to the invention can be used to characterise,quantify and map a nitrogenous base, a nucleic acid or a nitrogenousbase of a nucleic acid fixed on a support. It is characterized in thatit consists of characterising, quantifying and mapping the said nucleicacid or the said nitrogenous base by a mirage effect method.

In order to simplify the following description, the term “sample” isused to denote a nitrogenous base, a nucleic acid or a nitrogenous baseof a nucleic acid fixed on a support.

For example, nucleic acids and nitrogenous bases are disclosed in thebook entitled “Biochimie Générale” (General Biochemistry), J. H. WEIL,6^(th) edition, -MASSON, pages 279–288.

The term nucleic acid used in this description and in the appendedclaims denotes a chain of nucleotides connected to each other by 3′–5′phosphodiester bonds. The nucleotides are phosphoric esters ofnucleosides, and the nucleosides are derived from the bond between apuric or pyrimidic nitrogenous base with a ribose or a deoxyribose. Thenucleic acid is a ribonucleic acid (RNA) when the nucleotides formingthe nucleic acid contain ribose, and a deoxyribonucleic acid (DNA) whenthe nucleotides forming the nucleic acid contain deoxyribose. Thenitrogenous bases are usually adenine (A), guanine (G), uracile (U) andcytosine (C) when the nucleic acid is an RNA and adenine, guanine andthymine (T) and cytosine when the nucleic acid is a DNA.

The process according to this invention can be used to characterized,quantify and map RNA or DNA nucleic acids, and their nitrogenous basesand their derivatives.

For example, derivatives denote nucleic acids containing derivatives ofthe above mentioned nitrogenous bases, also called abnormal bases, forexample 5-hydroxymethylcytosine derived from cytosine.

This invention also describes a process for manufacturing a nucleic acidbiochip formed particularly of a solid support on which at least onenucleic acid synthesised in situ is fixed, the said process comprisingat least one synthesis and analysis cycle, including particularlyfirstly coupling of a nitrogenous base for in situ synthesis of the saidnucleic acid fixed on the support, and secondly an analysis to controlcoupling of the said nitrogenous base, the said analysis being madeusing a characterisation, quantification or mapping process according tothis invention.

Techniques for the in situ synthesis of nucleic acids are describedparticularly in the above mentioned books disclosing the manufacture ofbiochips, for example in Caruthers, Science, October 1985, page 281 andsubsequent pages. Obviously, coupling of the nitrogenous basecorresponds to formation of the 3′–5′ phosphodiester bond mentionedabove between the nucleic acid being synthesised and the nucleotidecontaining the nitrogenous base to be coupled.

It is important to note that the thin nucleic acid layers are usuallyconsidered as being non absorbent. This is disclosed particularly in“Ellipsometric and interferometric characterization of DNA probesimmobilized on a combinational assay”, Gray et al., Langmuir 1997, 13,2833–2842.

Despite this, these inventors have been interested in mirage effectmethods, also called photothermal methods.

All these methods use a light source, called a pump beam that is usuallya laser modulated at a given frequency, for excitation of the sample forwhich absorption is to be measured. Part of the incident light energy isabsorbed by the sample. The proportion of absorbed energy is fixed bythe absorption spectrum for the sample and the emission spectrum of theexcitation source. Part of the absorbed energy generates a localtemperature gradient that generates an index gradient.

Photothermal methods consist of detecting this index gradient.

The inventors have ingeniously demonstrated that the photothermaldeflection method is one of the photothermal methods, or mirage effectmethods, that can be used in this invention.

The photothermal deflection method consists of measuring the deviationof a light beam, called a probe beam passing through the area in whichthe index gradient is located. In other words, it consists of measuringthe deviation of the probe beam due to the temperature rise in anabsorbent sample by means of the pump beam. This photothermal deflectiontechnique was applied to surface analysis such as absorption mapping,thermal parameter imagery, but not to characterize, quantify and map anucleic acid or a nitrogenous base of a nucleic acid fixed on a support.

A complete presentation of the photothermal methods, sufficient toimplement this invention, may be found for example in the book entitled“Photothermal Spectroscopy Methods for Chemical Analysis, S. E.Bialkowski, vol. 134 in Chemical Analysis: a Series of Monographs onAnalytical Chemistry and its Applications, Wiley”.

Therefore, in the case of a photothermal deflection method, the nucleicacid or the nitrogenous base of the nucleic acid is illuminated by lightoriginating from an excitation source, and a probe beam is used todetect or measure the absorption, deviation or reflection of lightoriginating from the excitation source by the nucleic acid or by thenitrogenous base.

FIG. 1 appended contains a graph that shows the variation of theabsorption coefficient of the A, T, C, G and U bases as a function ofthe wave length, in order to determine the measurement wave lengths forthe process according to this invention. In this figure, the abscissarepresents the wave length λ in nm and the ordinate represents the molarabsorption coefficient (M.A.C.) (×10⁻³). Reference 60 indicates theabsorption spectrum of adenine (A), and references 62, 64, 66 and 68indicate the absorption spectra of thymine (T), cytosine (C), guanine(G) and uracile (U) respectively. These spectra can also be used tochoose the optimum sensitivity lengths of the DNA or RNA.

According to the invention, the excitation source may for example be acoherent source or an incoherent source.

The role of the pump beam is defined above. For example, it mayoriginate from a pulsed laser, or a continuous intensity modulated laserfor which the emission wave length is within the absorption band ofnucleic acids. In the case of nucleic acids, the orders of magnitudes ofthe layer thicknesses are usually a few nanometers.

Therefore according to the invention, the pump beam may be a coherentlight, for example a beam of a laser chosen among an argon laser with awave length of 275 nm, or a solid laser, for example quadrupled YAG witha wave length of 266 nm. According to the invention, absorption may bedetected or measured within a spectral range from 200 to 300 nm.

According to the invention, the pump beam may also be incoherent light,for example polychromatic light, if the emission spectrum of the sourcecan give a sufficiently strong signal for detection. For example,incoherent light may originate from a mercury vapour lamp.

The probe beam is preferably directed close to the portion of the sampleilluminated by the pump beam. Furthermore, the probe beam may beidentical to or different from the pump beam.

According to the invention, the probe beam preferably has a wave lengththat is not absorbed by the substrate or by the nucleic acids present.The probe beam is preferably a laser beam. For example, it may have awave length of between 400 to 700 nm. This facilitates alignment withrespect to the sample, because the wave lengths are within the visibledomain. For example, it may be derived from a helium-neon laser at 633nm.

The relative position of probe and pump beams defines the configurationused. For example, the probe beam may pass through one or several of thefollowing media—nucleic acids, the solid support or the surroundingmedium, for example a liquid or air. The orientation of the probe beamwith respect to the pump beam may be chosen at will, for example as afunction of the mechanical size and/or to optimise the sensitivity bysearching for the maximum absorption as a function of the angle ofincidence.

According to the invention, the probe and pump beams can intersect. Theposition of the intersection point, if any, can also be fixed at will,particularly as a function of the search for the optimum sensitivity. Ingeneral, the intersection point is located at the maximum thermalgradient.

According to the invention, the probe and pump beams may be arranged ina transverse configuration or in an approximately collinearconfiguration. In the transverse configuration, the probe and pump beamsintersect and are perpendicular to each other. This configuration isshown diagrammatically in FIG. 2 appended. In the approximatelycollinear configuration, the pump and probe beams intersect but arealmost collinear. FIG. 3 appended is a diagrammatic representation ofthe collinear configuration.

In these figures, reference 1 indicates the pump beam, reference 3 theprobe beam in the transverse configuration, reference 3 indicates theprobe beam in the approximately collinear configuration, reference 7indicates a laser, reference 9 a detector such as a detector with fourquadrants and reference 11 indicates the sample consisting of the solidsupport on which the nucleic acids are fixed.

The reflection or refraction of the probe beam may be detected using amulti-element photodiode, for example a detector with two or fourquadrants, a strip or a matrix, or using a simple photodiode, eitherpartially covered by a cache or a blade, or only receiving part of theprobe beam.

In the case of a simple photodiode, another detector may be necessary todissociate variations in the absorption of nucleic acids from variationsin the power of the pump beam, if any.

FIG. 4 shows a diagrammatic illustration of different configurations fordetection of the deviation of the probe beam; In this figure, -A-diagrammatically represents a bi-quadrant detector 13 and a spot 15formed by the probe beam on the detector, -B- represents a 4-quadrantdetector, -C- represents a matrix detector, -D- represents a simplephotodiode partially covered by a cache 17, and -E- represents anoff-centred photo detector.

In order to obtain sufficient sensitivity, the deflection of the probebeam induced by the nucleic acid, or the nitrogenous base of the nucleicacid is preferably distinguished from parasite variations due to themedium, such as temperature variations in the laboratory. In order toachieve this, the pump beam may be marked in time, either by amodulation if it is continuous and by its pulse operation. Control ofthe modulation frequency or the possibility of obtaining a referencesignal is a means of preferably detecting reflection due to atemperature rise caused by partial absorption of the pump beam.

Regardless of the configuration of the beams used, the informationobtained is local and applies to an area around the impact point of thepump beam on the trace nucleotides or the nitrogenous base.

The size of this area may be fixed by experimental parameters such asthe size of the pump beam at the impact point on the sample or itsmodulation frequency, and by the thermal behaviour of the support, thesample and the ambient medium. This is due particularly to thedistribution of heat in the different media.

Since the information is local, the detection instrument may be coupledto a support displacement system onto which the nucleic acids are fixedrelative to the pump beam. The assembly can then be used to comparedeviation values of the probe beam from one point on the support toanother, and in particular the signal may be represented in the form ofa map.

Whenever necessary, the deviation signal may be converted into anabsorption value, for example by means of a reference sample or acontrol sample. This control sample, the absorption of which is knownand stable, may be the subject of a photothermal deflection measurementunder the same experimental conditions as for nucleic acids ornitrogenous bases. For example, the value obtained can be used tocalculate a conversion coefficient for the electrical signal measured atan absorption level.

The model of the interaction of the probe beam with the pump beam showsthat losses can be measured by absorption of the sample. As we havealready seen, the instrument can be calibrated by measuring thedeflection signal of a reference sample for which absorption is known inadvance. If absorptions are low, it can be shown that the photothermaldeflection signal is proportional to absorption of the sample.Therefore, it is possible to work relatively with respect to thereference sample.

With the process according to this invention, losses by absorption of afew ppm (10⁻⁶) can be measured. The term “losses” refers to the ratiobetween the power absorbed in the material and the incident light power.One advantageous embodiment of this invention may be to use absorptionof the pump beam in P polarisation, in other words parallel to the planeof incidence of trace nucleotides in order to optimise the detectionsensitivity. In some cases, absorption in P polarisation passes througha maximum at the Brewster angle of the substrate, for example 56.6° forglass.

Each base has a specific absorption spectrum that can be used. Accordingto this invention, we will follow the variation of the photodeflectionsignal as a function of the number of bases.

As a first order, the absorption coefficient of an elementary layer iswritten:A=αe=(4πk/λ).e

-   -   where A denotes the absorption of an elementary layer, α denotes        the absorption coefficient, e denotes the mechanical thickness,        k denotes the thin layer extinction coefficient and λ is the        wave length of the pump beam.

After the growth of N bases i, we obtain simply:$A = {{\sum\limits_{i = 1}^{i = N}{\alpha_{i}e_{i}}} = {\sum\limits_{i = 1}^{i = N}{\left( {4\pi\;{k_{i}/\lambda}} \right) \cdot e_{i}}}}$

In the biology domain, this coefficient can also be expressed in thefollowing form:$A = {\sum\limits_{i = 1}^{i = N}{\ln\mspace{14mu} 10ɛ_{i}c_{i}}}$

ε₁ and c_(i) denote the molar extinction coefficient (1.mol⁻¹.cm⁻¹) andthe concentration (mol.1⁻¹) respectively, for each base.

For guidance, extinction coefficients are of the order of 10⁻² to 10⁻³and measurable absorptions are of the order of a few hundred ppm.Therefore, they are weak optical signals.

As a first order of magnitude, steps i are additive on the previoussteps. Therefore, the signal is easy to interpret and can be used tomonitor the variation of the growth of bases with the steps for in situsynthesis of trace nucleotides.

According to the invention, the support may for example be made ofglass, or oxidised silicon, plastic or a gel. For example this supportcould be a plane support or it could contain micro-cavities, for examplemicro-bowls.

According to the invention, the first nucleotide may be fixed on thesupport for in situ synthesis by conventional chemical reactions, chosenfirstly depending on the support and secondly so as to fix thenucleotide on the preferred support by a covalent bond. These chemicalreactions are disclosed, for example, in Chemistry Letters, 1998, p.257–258 and Analytical Biochemistry 247, p. 96–101, 1997.

This invention also provides a device for implementation of the processaccording to this invention, particularly when the method is aphotothermal deflection method.

The device comprises the following elements:

-   -   a means of positioning the sample comprising a support on which        the nucleic acids are fixed,    -   a means of illuminating the sample,    -   a means of detecting and/or measuring the absorption, deviation        or reflection of light by the sample when it is illuminated by        the said illumination means, and    -   a means of positioning the said illumination means and the said        detection and/or measurement means.

According to the invention, the means of positioning the sample may beany known means of precisely displacing the micrometric translation androtation plates, for example made by MicroCôntrole or Spinder Hoyer.These means may be motor driven to enable automation, particularly formapping.

According to the invention, the means of illuminating the sample anddetecting absorption of light may be chosen particularly as a functionof the support and the nucleic acids to be detected. The means ofilluminating the sample may for example consist of a pump beam asdefined above. The means of detecting absorption may comprise a probebeam and means of detecting refraction or reflection of a probe beam.These means are described below and in the following examples.

According to the invention, the positioning means and the illuminationand detection means mentioned above may be means such as those mentionedabove for positioning the sample.

Therefore, the invention is innovative particularly due to the fact thata photothermal technique has never been used to characterise, quantifyand map a nitrogenous base, a nucleic acid or a nitrogenous base of anucleic acid fixed on a support. More generally, no method has ever beenused based on the measurement of the absorption variation for this typeof analysis on support.

In particular, the process according to the invention has the advantagethat it does not necessitate any marking step or marker. For example, itmay advantageously be used for the manufacture of nucleic acid biochips.Characterisation, quantification and mapping of each nitrogenous base,for example at each in situ synthesis step of a DNA biochip or an RNAbiochip, can be used to precisely monitor each step of the saidsynthesis, and consequently to control the density, the uniformity ofthe density and the quality of the manufactured biochip and possibly tocorrect any errors that occur during the synthesis.

This was not possible with techniques according to prior art. Biochipsobtained using the process according to this invention are precise,homogeneous and easily reproducible.

The process according to this invention can be used to optimise the insitu synthesis process for trace nucleotides for the manufacture ofbiochips and to obtain a coupling efficiency equal to or close to 100%;This use will be illustrated in the following examples.

Other elements and advantages of the invention will become clear afterreading the description and the examples given below with reference tothe appended drawings, obviously given for illustrative purposes and inno way limitative.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the invention will become clear afterreading the following description, in reference to the appendeddrawings, in which:

FIG. 1 is a graph that shows the variation of the molar absorptioncoefficient of G, C, T and A nitrogenous bases, as a function of thewave length;

FIG. 2 is a diagram illustrating a photothermal deflection in transverseconfiguration for the analysis of a sample to be tested using theprocess according to this invention;

FIG. 3 is a diagram illustrating a photothermal deflection inlongitudinal configuration for the analysis of a sample to be testedusing the process according to this invention;

FIGS. 4 A, B, C, D and E diagrammatically illustrate differentconfigurations for detection of the deviation of the probe beam;

FIG. 5 is a diagrammatic illustration of a measurement of the absorptionof a sample using the photothermal deflection method in transverseconfiguration according to the invention;

FIG. 6 is a diagram illustrating a device for implementation of theprocess according to this invention;

FIGS. 7 and 8 diagrammatically show the method of determining the“image” after each step in the synthesis of a probe using the processaccording to this invention;

FIG. 9 shows the variation with time of photothermal signals obtained onsilica substrates according to this invention.

EXAMPLES Example 1 Measurement Method

The system used in this example according to the invention is based onphotothermal deflection in transverse configuration.

FIG. 5 appended is a diagrammatic illustration showing the principle ofa measurement of the absorption of a sample composed of a nucleic acidor a nitrogenous base of a nucleic acid fixed on a support by thephotothermal deflection method according to the invention.

In this figure, the pump beam 19 is derived from a continuous argonlaser at 275 nm (COHERENT INOVA 40) (trade name) (not shown), it isfocused on the sample 11 by means of a spherical mirror (not shown), thediameter of the spot (not shown) is about 70 microns at the surface ofthe sample 11. The wave length of the pump beam is chosen to enabledetection of nucleic acids. The power of the pump beam is 300 mW at thelaser output.

The probe beam 21 is a probe beam from a helium-neon laser at 633 nm.The wave length of this probe beam is indifferent. According to theinvention, a wave length very different from the wave length of the pumpbeam can give a better signal to noise ratio.

The deflection of the probe beam 21 is detected by means of a fourquadrant detector -B- (see FIG. 3) followed by amplification andsubtraction electronics (not shown). Reference 21 a indicates the probebeam deviated by photothermal deflection. The angle θ indicates theangle of incidence of the pump beam 19 from the normal 20 to the sample(shown in chain dotted lines) and reference 19 a indicates the pump beam19 reflected on the sample.

An interference filter (not shown) selecting the wave length of theprobe beam may be placed in front of the four quadrant detector in orderto avoid the influence of parasite light originating from the modulatedpump beam.

In the device according to this invention, the probe laser, the fourquadrant detector and the associated electronics can form an integralpart of a commercial measurement cell, for example made by the ALISCompany. The signal output from this cell is sent to a synchronousdetection.

The pump beam may be modulated by means of a disk with a mechanicalslit, also called a mechanical chopper in the following, the frequencyof which is adjustable. The chopper control signal is used as areference for synchronous detection. The frequency is 157 Hz. Themeasured signal is obtained at the output from the synchronous detection(amplitude of the deviation signal at the pump beam modulationfrequency).

The sample and the two beams are positioned with respect to each otherby micrometric translation and rotation plates (made by Micro-Contrôle),some of which are motor driven to enable automation for mapping, forexample of a biochip, and in some phases of the adjustment. Theadjustments are made automatically to maximise the deflection signal ina plane orthogonal to the sample containing the probe beam. Duringmapping, a corrective displacement is made if necessary in a directionorthogonal to the map scanning axes in order to guarantee that therelative position is correctly maintained during mapping. Thiscorrective displacement is determined automatically in a preliminarymeasurement step. The angle of incidence of the pump beam with respectto the normal to the sample and the orientation of the cell with respectto the sample, may be adjustable. The relative positions andorientations of the probe and pump beams may also be adjustableindependently.

A diagram of the device according to the invention is shown in FIG. 6appended. In this figure, a shutter, not shown on the diagram,interrupts the pump beam during the displacement phases and restores itduring a clearly defined time period after a waiting time chosen toallow the set-up to reach a steady state after a displacement. Thereference 23 indicates an argon laser at 275 nm, reference 25 indicateslaser beam positioning mirrors, reference 27 indicates a mechanicalchopper, reference 29 indicates the laser beam after it has passedthrough the chopper, reference 31 indicates a focusing mirror, reference35 indicates the measurement sample, reference 36 indicates the probehelium-neon beam and reference 37 indicates the four quadrantphotodiode.

For example, the position of the sample may be made repeatable by usinga self collimating sight that is not shown on the diagram. The entiremeasurement device may be controlled by a workstation that controlsdisplacements and acquires deviation signals in two orthogonaldirections.

In this embodiment, the deviation is measured along a direction parallelto the plane of the sample and along a direction orthogonal to it. Thissecond direction forms the useful signal. The electronic signal suppliedby the synchronous detection may be used as such by comparison betweendifferent points on the sample.

It can also be converted into an absorption value by making a referencemeasurement on a sample considered to be stable in time and under alaser flux, the absorption of which can be measured with aspectrophotometer and which is located in the linearity range of thephotothermal deflection measurement bench.

Example 2 In Situ Synthesis of Oligonucleotides on Silica Substrate

The silica substrates used are of the Suprasil type (SESO Company), theyhave a diameter of 50 mm and are 3 mm thick.

1—Surface Hydroxylation

A solution containing 2 g of soda NaOH/6 ml of deionised water/8 ml of95% ethanol is prepared and the substrates are incubated in it for 2hours. The samples are then rinsed with deionised water and dried with anitrogen blower. This step enables the creation of hydroxyl groups atthe surface of the silica (see formula (I) below).

2—Silanisation

2 ml of 3-glucydoxypropyltrimethoxy silane (98%) is put into solution in7 ml of toluene and 0.6 ml of triethylamine. The samples are put intothis solution for one night at 80° C. They are then dried with acetoneand nitrogen using a blower and are annealed at 110° C. for 3 hours. Thechemical silanisation reaction may be shown diagrammatically as follows:

3—Opening of the Epoxide Function

A solution containing 30 ml of hexaethylene glycol and 18 μm ofsulphuric acid is used to enable opening of the epoxide bond and thus tofix hexaethylene glycol. The opening of the epoxide function may beshown diagrammatically as follows:

4—Synthesis of Oligonucleotides

The synthesis of oligonucleotides by the phosphoramidite method isdisclosed in document Caruthers, Sciences, October 1985, page 281. itcomprises the following steps:

Detritylation, coupling, capping and oxidation.

Detritylation:

The sample is put into 2 ml of a solution containing 3% oftrichloracetic acid in dichloromethane (DMT Removal batch 2257-1 Roth)for 2 minutes while stirring. It is then rinsed in dichloromethane andthen in acetonitrile and is dried under a pressurized nitrogen jet.

Coupling:

2 ml of a solution containing 25 mg of2′-deoxy-5′-O-dimethoxytrityl-3′-O— (βcyanoethyl N,N-diisopropylamino)phosphoramidite is mixed with 150 μm of tetrazole. The sample isput into this solution and stirred under argon for 10 minutes. It isthen rinsed in acetonitrile and then dried by a pressurised nitrogenjet.

Capping:

A solution is prepared containing 1 l of CAP and 1 ml of CAP2(CAP1=acetic anhydride/lutidine/THF, CAP2=1-methylimidazole/THF). Thesample is put into this solution for 2 minutes. It is then rinsed inacetonitrile and dried by a pressurised nitrogen jet.

Oxidation:

The sample is put into a solution of 4 ml of Reagent Oxidation(iodine/water/pyridine/THF batch 2254.2 Roth) while stirring for 1minute. It is then rinsed in acetonitrile in dichloromethane and then inacetonitrile, and is dried by a pressurised nitrogen jet.

These steps are repeated N times, where N is the number of mers of theoligonucleotide to be synthesised. The coupling step determines thenature of the base of each nucleotide (A, C, G, T or U).

Example 3 Absorption Measurement for Different Lengths of Oligomers

This example was made on silica supports as prepared in example 2, witha thickness including 1mer, 2mers and 8mers and with sequences composedof T bases only.

The purpose of this example is to show that the process according tothis invention can be used to differentiate the growth of anoligonucleotide base by base.

We made four different samples called Sample 1 to sample 4 using themethods described above. These samples are as follows:

-   -   Sample 1: silica/NaOH treatment/silanisation/HEG,    -   Sample 2: silica/NaOH treatment/silanisation/HEG/synthesis of a        monomer comprising a T base,    -   Sample 3: silica/NaOH treatment/silanisation/HEG/synthesis of a        dimer comprising a T base on each nucleotide,    -   Sample 4: silica/NaOH treatment/silanisation/HEG/synthesis of a        octomer comprising a T base on each nucleotide.        1) Measurement Parameters:

Measurements were made using an argon laser.

Wave length 275 mm; 140 mW sample power; chopping frequency F=157 Hz;angle of incidence 45°; detection on two quadrants.

2) Measurement:

Mapping on 1 mm² at the centre of the samples in steps of 0.1 mm.

For each point X, Y, acquisition of absorption=f(t), where 0<t<4 secondsin time steps of about 100 ms. The acquisition takes place as follows:

-   -   for 0<t<1 second by mirage effect: the shutter is closed,    -   at t=1 second, the shutter is opened,    -   for 1≦t≦4 seconds: absorption measurement.

The results are given in table I below:

TABLE I Maximum absorption value (Amax) for each sample Sample No.Sample nature Amax at 275 nm Sample 1 Silane + HEG 225 ua Sample 2Silane + HEG + 1 T oligo 280 ua Sample 3 Silane + HEG + 2 T oligo 360 uaSample 4 Silane + HEG + 8 T oligo 730 ua

-   -   where ua: absorption units.

FIG. 9 appended shows the variation of the photothermal signals as afunction of time obtained on silica substrates comprising glycolhexaethylene (HEG), a monomer with one T base (HEG+T), a dimer with twoT bases (HEG+2T) and a octomer with eight bases T (HEG+8T).

These curves show the reference (no nitrogenous base), the signal forone T base, two T bases and eight T bases, respectively.

Example 4 Determination of an Image at Each Step in the Synthesis of anOligonucleotide

The “image” of each synthesis step is determined by comparison with theprevious steps. FIGS. 7 and 8 appended diagrammatically show teststructures. In these figures, the test structures are showndiagrammatically by squares, or pads, corresponding to areas for example100×100 μm² in which the optical response will be measured for thesubstrate (ref), the four bases grafted on the substrates (A, C, G andT) separately, the grafted dimers corresponding to different possiblearrangements in the final oligonucleotide (AT, AC, . . . , TT).

With these reference pads, we measured the signature of the absorptionof each base for the measurement wave length(s). With these absorptionmeasurements, the number of G, C, T, A bases present in theoligonucleotide probe was measured by means of simple subtractions shownin FIG. 7.

As shown in FIG. 8, the signal of each base was calculated regardless ofits environment, by making successive subtractions. Thus, in the firstline reference 80, the signals of A were determined with respect to A,C, G and T respectively, and in the second line reference 82, thesignals of C were determined with respect to A, C, G and T respectively,and in the third line reference 84, the signals of G were determinedwith respect to A, C, G and T respectively, and in the fourth linereference 86, the signals of T were determined with respect to A, C, Gand T respectively.

Example 5 Process for Synthesis of a Biochip According to the Invention

As presented in the description and the above examples, this inventionis intended to provide a process for the analysis of each coupling stepin the in situ synthesis of oligonucleotides on structured substrates.

The analysis is non-destructive.

However, the inventors have observed that the sample may be damagedabove a given power density transmitted to the sample, of the order of 1kW/cm².

1. A method for detecting an unmarked molecule(s) fixed on a support,wherein said unmarked molecule(s) is a nitrogenous base or a nucleicacid that has not been marked with a dye comprising: illuminating themolecule(s) by light originating from an excitation source or pump beamhaving a wavelength ranging from 200 to 300 nm, and detecting ormeasuring the absorption, deviation or reflection of light originatingfrom the excitation source by the molecule(s) using a probe beam,thereby detecting said unmarked molecule(s).
 2. The method of claim 1,wherein said support is glass.
 3. The method of claim 1, wherein saidsupport is oxidized silicon.
 4. The method of claim 1, wherein saidsupport is plastic.
 5. The method of claim 1, wherein said support isgel.
 6. The method of claim 1, wherein said molecule(s) is a nitrogenousbase.
 7. The method of claim 1, wherein said molecule(s) is anitrogenous base selected from the group consisting of A, T and U. 8.The method of claim 1, wherein said molecule(s) is a nitrogenous baseselected from the group consisting of G and C.
 9. The method of claim 1,wherein said molecule(s) is an oligomer comprising two or morenitrogenous bases.
 10. The method of claim 1, wherein said molecule(s)is an oligomer or a nucleic acid which has been synthesized on saidsupport.
 11. The method of claim 1, comprising detecting or measuringthe absorption, deviation or reflection of light originating from theexcitation source by a nitrogenous base of a nucleic acid using a probebeam.
 12. The method of claim 1, wherein said molecule(s) is aribonucleic acid.
 13. The method of claim 1, wherein said molecule(s) isa deoxyribonucleic acid.
 14. The method of claim 1, wherein saidmolecule(s) is illuminated by a pump beam which is coherent.
 15. Themethod of claim 1, wherein said molecule(s) is illuminated by a pumpbeam which is incoherent.
 16. The method of claim 1, wherein saidmolecule(s) is illuminated by a pump beam which is an argon laser with awave length of 275 nm.
 17. The method of claim 1, wherein saidmolecule(s) is illuminated by a pump beam which is a solid laser with awave length of 266 nm.
 18. The method of claim 1, wherein the probe beamhas a wavelength ranging from 400 to 700 nm.
 19. The method of claim 1,wherein the probe beam is different than the pump beam.
 20. The methodof claim 1, wherein the probe and pump beams intersect at the maximalthermal gradient.
 21. The method of claim 1, wherein the probe and pumpbeams are arranged in a transverse configuration.
 22. The method ofclaim 1, wherein the probe and pump beams are arranged in anapproximately collinear configuration.
 23. The method of claim 1,wherein the probe beam passes through the solid support.
 24. The methodof claim 1, wherein the probe beam passes through the molecule(s). 25.The method of claim 1, wherein the reflection of the probe beam isdetected.
 26. The method of claim 1, wherein the refraction of the probebeam is detected.
 27. The method of claim 1, wherein the absorption oflight from the pump beam by the molecule(s) is measured.
 28. The methodof claim 1, wherein the absorption of light from the pump beam ismeasured within a spectral range of 200 to 300 mm.
 29. The method ofclaim 1, wherein the absorption of the pump beam parallel to themolecule(s) (P polarization) is measured to optimize the detectionsensitivity.
 30. A method for synthesizing an oligonucleotide or nucleicacid on a solid substrate, comprising: coupling a nitrogenous base to asolid substrate, at least one cycle of coupling one or more additionalnitrogenous bases to the nitrogenous base fixed to the solid substrateor to the oligonucleotide fixed to the solid substrate to form anoligonucleotide or nucleic acid, and detecting the oligonucleotide ornucleic acid synthesized on said substrate by: illuminating themolecule(s) by light originating from an excitation source or pump beamhaving a wavelength ranging from 200–300 nm, and detecting or measuringthe absorption, deviation or reflection of light originating from theexcitation source by the molecule(s) using a probe beam.
 31. The methodof claim 30, wherein said synthesizing comprises multiple cycles ofcoupling a nitrogenous base to a nitrogenous base or oligonucleotidefixed on the support, and said method comprises detecting thenitrogenous based or the oligonucleotide fixed on the support after eachsynthesis cycle.
 32. A method for making a biochip which comprises anoligonucleotide or nucleic acid fixed to a support, comprising:synthesizing on the surface in situ an oligonucleotide or nucleic acid,and detecting said oligonucleotide or nucleic acid on said surface by:illuminating the molecule(s) by light originating from an excitationsource or pump beam having a wavelength ranging from 200–300 nm, anddetecting or measuring the absorption, deviation or reflection of lightoriginating from the excitation source by the molecule(s) using a probebeam.
 33. The method of claim 32, wherein said synthesizing comprises atleast one cycle of coupling a nitrogenous base to a nitrogenous base orto an oligonucleotide fixed on the support, and said method comprisesdetecting the nitrogenous base or the oligonucleotide fixed on thesupport after each synthesis cycle.